Bacterial Media Recipes

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When making media it is often useful to make it in a larger bottle than you plan to autoclave it in because some of the media may come out of the bottle on autoclaving. Always loosen the lid of a bottle when autoclaving the media, otherwise the bottle may break/explode.

Terrific broth is made in two separate solutions that are combined after sterilisation.

Media base solution:

Measure out 750ml of distilled H2O

Add:

12g Tryptone

24g Yeast extract

4ml Glycerol

Make the volume up to 900ml with distilled H2O

Sterilize by autoclaving

Allow the media to cool to room temperature

Measure 90ml of distilled H2O.

Phosphate Buffer Solution:

Measure 90ml of distilled H2O

Add 2.31 g KH2PO4 monobasic

Add 12.54 g K2HPO4 dibasic (for trihydrate 16.45 g)

Adjust the volume to 100 mL with distilled H2O

Sterilise by either filtration or autoclaving

Add the Phosphate buffer to the media base solution but only after the media has cooled to at least 60ºC.

DNA Storage Buffers

TE buffer: (for standard DNA manipulations)

10mM Tris-HCl pH 8.0
0.1mM EDTA

Tip 1: DNA is quite stable in TE buffer at 4ºC. If stored in elution buffer or water then freezing at -20 ºC is advised.

Note 1: TE can reduce some enzymatic reactions, for example, DNA sequencing reactions and occasionally ligations. Elute DNA to be ligated or sequenced in either elution buffer or water.

Elution Buffer (Used by many of the kits from major suppliers)

10mM Tris-Cl, pH 8.5

Note: Elution buffer is basically TE without the EDTA which is the component that can disrupt enzymatic reactions. DNA is more stable in this than in water.

Nuclease Free water

To make nuclease free water, fill a small clean lab bottle with deionised water filtered through a 0.22µm pore filter using a syringe and then autoclave. Replace every few weeks/months to avoid contamination.

Note 1: Tissue culture grade nuclease free water is available from many suppliers. It is not that expensive (<£2) but you can just make it yourself. You will only be using small amounts so if you do buy it will last you a while if it doesn’t become contaminated.

Potential Hazard: Powders can be sensitising and you can develop allergies to them. Avoid inhalation of aerosolised powder.

Protocol

Measure out the amounts above and add to a 1 litre bottle with a good working lid (see note below). Fill with clean distilled water (ideally at least 18 megaohms). Tighten lid and shake to mix the liquid and powder, don’t expect to dissolve it all but simply to free the powder from the bottom and remove the major clumps. If not mixed properly the powder can bake on the bottom of the bottle. Undo the lid about half a turn; add some autoclave tape and then autoclave.

After autoclaving make sure the lid is tightened back up. Ideally the lid is tightened whilst the bottle is warm/hot as this will create a vacuum as the liquid cools. When cool add the correct amount of antibiotic (see below) and store at 4ºC. It can be left in the cold room for a few months but watch out for fungus contamination and a decline in antibiotic activity over time. To maintain sterility it is best to add the antibiotic and take aliquots out of the bottle in a class 1 or 2 laminar flow hood.

Notes and Tips:

Tip 1: Many lab bottles have a blue or clear plastic rim which can go missing or get burnt by flaming. These blue rims prevent the liquids running down the bottle when pouring and keep an air tight seal after autoclaving. Make sure the rim is intact.

Tip 2: 1 litre is quite a lot of media and antibiotic, scale down the recipe to suit your needs. I generally make 0.5 litres at a time.

Tris Acetic Acid EDTA

Tip 1: For larger DNA fragments (>10 kb) TAE is recommended as the image resolution may be slightly better. TAE has a lower buffering capacity compared to TBE.

Tip 2: For small DNA fragments (<1 kb) TBE is recommended as the image resolution is better. It has a higher buffering capacity than TAE.

Tip 3: Avoid using buffers more than 3 times. As time goes by the water evaporates and increases the concentration of the buffer components. For this reason don’t use too many new gels in old buffers as the concentrations will be different between the two and cause an imbalance in the conductivity.

Note 1: TAE heats up more than TBE which can cause damage to gels and equipment on regular long (>4 hr) runs.

Note 2: Some gel extraction kits recommend one buffer and may work more efficiently with that buffer. Check gel extraction kit manuals carefully. Most kits seem to have no buffer preference.

Agarose Gel Loading Dye

Note: Bromophenol blue can get everywhere. Be very careful with the powder as even small amounts can cause problems. Your lab collegues will not be happy if they find everything turns blue when they add water to it.